Biochemistry Pre-Lab Assignment

In the gel filtration step, we began with a slurry of Bio-Gel P-100 beads suspended in 20 mM phosphate buffer (Equilibration buffer) and an upright column with a stopcock. We added enough of the Bio-Gel P-100 beads to the column until we reached a height of 4.7 cm, making sure not to allow the column to run dry and that the top of the beads column is flat. We then added the sample, a mixture of Blue Dextran (2 mg/ml, 2 MDa), Hemoglobin (2 mg/ml), Bovine Serum Albumin (2 mg/ml), and Yellow Food Coloring (5 μl/ml, ~500 Da) (Sheffield, 37). This was followed by another ~1 ml of equilibration

In this lab, paper chromatography will be used to separate the components of known and unknown mixtures and then used to identify those components. Paper chromatography is a technique where a drop of solution containing a mixture will be placed on a piece of filter paper. One end of the filter paper will then be placed into a liquid solvent. The mixture will separate into its different components as the solvent moves up the filter paper. The filter paper is known as the stationary phase. The solvent is known as the mobile phase because it uses capillary action to move up the paper. The mixture will be separated into its different components because each component will have a unique chemical affinity for the paper and for the solvent. Theses

On a thin chromatography plate, five spots were placed ( as shown in table 2) and the plate was developed using chloroform/methanol. This was later visualized with dragendorff’s reagent under the UV light. All separated components were observed, identified and recorded.

Sephadex chromatography (gel-filtration chromatography) separates molecules on the basis of their size. It is a form of column chromatography in which the stationary phase consist out of cross-linked gel particles. The gel particles are usually in bead form and consist of agarose. Molecules of varying size move through a bed of permeable beads, diffusing into the beads. Small molecules are able to diffuse further into the pores of the beads, thus these smaller molecules move through the bed slowly. Large molecules do not enter or enter less and thus move through the bed faster or quickly. Sephadex chromatography may be used for the determination of molecular size, for the separations of specific components in a mixture, or for preparation of macromolecules.

In this paper I will explain how metabolism works and the thyroid gland function in the human body. First the two different types of metabolism: Anabolism, Catabolism and how the nutrition also affects the production of the thyroid hormone. Secondly, the function of the thyroid hormone, which organs regulate, consequences if the human body begins if it works to work to fast or too slow. Third, the different hormones that the thyroid gland produces and how it regulates the metabolism. Fourth, there is certain nutrients that help the thyroid gland to produce the hormone and also there is another ones that affect the production of the thyroid hormone. Last how the six different types of thyroid problems are found, the symptoms and how they

Explain the role of RNA polymerase inhibition in causing the poisonous effect of the death cap mushroom.

The students selected eluting flow through, E.coli flow through, and washing flow through samples from lab #6 that contained protein. For all samples, they mix 15 microliters of the selected sample with 4 microliters of 5x sample loading buffer and 1 microliter 20x reducing agent, and heat the samples at over 90 degree Celsius for 5 minutes. In the meantime, the students proceed to prepare 40 ml of 15% resolving gel solution using 9.68 ml of H2O, 20 ml of 30% Acrylamide/bis solution, 10 ml of 4 x resolving gel buffer, 300 ul of 10% APS, and 20 ul of TEMED. First they prepared the resolving gel buffer, to do this they mix 9.09g of 1.5 M Tris-HCl, 0.2g of 4% SDS, and approximately 30 ml of H2O. When all the components where mix, they checked the pH and verify that the solution has a pH of 8.8, if that was not the case they proceed to add HCl or Tris, depending whether the solution was more acidic or base, until the desire pH was reach.

Bio Pre Proposal I. OBJECTIVE: To have the ability to identify gram positive and gram negative cells and understand how the staining affects them. II. HYPOTHESIS: if a smear of both Escherichia coli and Staphylococcus epidermidis are stained, then gram negative and gram positive stains should result respectively and be identifiable under the microscope due to the structure of the cell walls. III. PROCEDURE: 1.

Column one was a 30m x 0.25mm-i.d coated with 0.5 μm 5% diphenyl and 95% dimethyl polysiloxane (Rtx®-5). Column two was a 30m x 0.25mm-i.d coated with 0.5 μm midpolarity phase consisting of 50% phenyl and 50% dimethyl polysiloxane (Rxi®-17sil). Column three was a 30m x 0.25mm-i.d, capillary coating with 0.5 μm film of 100% trifluoropropyl methyl polysiloxane (Rtx®-200). The separation was performed using the same temperature program on all these stationary phases. The temperature program used for separation was consisted of an initial temperature hold at 80 °C for 1.0 min, ramped up to 300 °C at a rate of 30 °C/min, held at 300 °C for 0.5 min then ramped to 340°C at a rate of 5.0 °C/min and held at 340 °C for 5.0 min with a total run 21 min. (TP1). Column four was a 30m x 0.25mm-i.d, capillary coated with 0.5 μm film of midpolarity phase consisting of 35% phenyl and

In this lab, paper chromatography will be used to separate the components of known and unknown mixtures and then used to identify those components. Paper chromatography is a technique where a drop of solution containing a mixture is placed on a piece of filter paper. One end of the filter paper is then placed into a liquid solvent. The mixture will separate into its different components as the solvent moves up the filter paper. The filter paper is known as the stationary phase. The solvent is known as the mobile phase because it uses capillary action to move up the paper. The mixture will be separated into its different components because each component will have a unique chemical affinity for the paper and for the solvent. Theses affinities

30uL of the LB Buffer added to 30uL of each saved samples). Similar addition of LB Buffer (10uL) was made with the Protein Standards Ladder (10uL). After the addition of the LB Buffer to the appropriate samples, it was placed on a 95oC hot plate for 8 minutes (note: it was instructed to be for 5 minute). This was followed by the preparation of the gel setup (i.e. removing the tape at the bottom of the precast gel and adding the gels at the back and front of the apparatus gel). To this setup, 50uL of 10X SDS running buffer + 450uL of deionized water (making it a 1X Buffer) to the outer chamber was added. In the inner chamber, 150uL of 1X SDS (1X was made from the 10X SDS buffer, the same way by diluting it with a 1:9 ratio of deionized water) running buffer was added. With such setup and the 5 samples that was heated in 95oC, the lanes—starting with lane 1—were pipetted in the following order: 10uL of Protein Standards sample (lane 1), 20uL of “+IPTG with Elution Buffer” (lane 2 and 3), 20uL of “-IPTG with Elution Buffer” (lane 4 and 5), 20uL of “+IPTG Supernatant” (lane 6 and 7), and 20uL of “-IPTG Supernatant” (lane 8 and 9). After loading the gel in such manner, the start of running the gel was done at 200V (making note of it being stable initially), and it was stopped till the bromophenol blue bands reached ~1cm

Proteins, or nucleic acids, are isolated and separated within a gel. The gel is cast in a thin, welled slab. In this particular experiment, the gel will be

Gel-Filtration Chromatography is a commonly used method used in order purify a protein from a mixture, by means of separations. Different biomolecules differ in size, or their molecular weight. In the gel matrix inside the chromatography column, there are gel beads which are porous to allow certain sized molecules to enter. The molecules that are able to enter the pores of the gel, are held in stationary phase and will elute from the column later on, these are usually smaller, to medium sized molecules. Larger molecules that are not able to fit in the pores will elute out of the column first, they are involved in mobile phase where they just go straight through the column without interacting with the gel beads. Smaller molecules will have a higher elution volume, while the larger molecules will have a lower elution volume. The volume to elute the protein is inversely proportional to the molecules size.

In modern chromatographic separations, the stationary phase comes in the form of media, either pre-packed in a chromatography column, or added by the user to the separation apparatus. The choice of media depends on the type of molecule being separated, the means of separation, the purpose and scale of separation, and the desired purity of the separated sample.

Column chromatography consists of solid-liquid phase partitioning. Several steps are involved in order to perform a separation using this technique. The first step is selecting a column. The size of the sample which will be separated or purified will indicate what size the column should be. The difference in Rf values is also a